Rickettsial diseases are of major, often unrecognized, importance to the public health worldwide. RMSF, the most severe and widespread rickettsiosis in the U.S., continues to kill more than 5% of patients because of diagnostic difficulty and lack of means for prevention. Boutonneuse fever has a higher incidence and significant mortality in Africa, Europe, and Asia. The long-term goal of this project is to analyze the structural and molecular Composition of SFG rickettsial antigens and to determine which components stimulate protective immunity. This proposed research is designed to extend our knowledge of the immunobiology of the SFG rickettsiae by the molecular characterization of the rickettsial protein antigens and to identify the critical immune effector mechanisms that must be stimulated by immunization to give protective immunity. We propose to dissect the important functional and molecular interactions of these antigens with the immune system, particularly for T-lymphocyte directed cell-mediated immunity in order to elucidate the interaction of the immune system with intraendothelial events. We plan to emphasize the correlation of immunodominant T- lymphocyte epitopes with investigation of the mechanisms of the killing and clearance of rickettsiae from their intracellular location in the cytosol of endothelial cells. Our specific aims are 1) to identify the human T-lymphocyte epitopes of Rickettsia rickettsii and the protective and cross protective mouse T- lymphocyte epitopes of R. conorii and R. rickettsii, 2) to investigate the mechanism by which cytokines induce the killing of rickettsiae in human target cells, 3) to determine the role of cytotoxic T-lymphocytes in the clearance of rickettsial infection, and 4) to explain why killed rickettsial antigen and purified subunit vaccine confer less effective immune protection than antigen expressed by living organisms. The methods that will be employed include establishment of representative populations of human and mouse antiR. rickettsii T-lymphocyte lines and clones including CTL clones, production of recombinant polypeptides of the major surface proteins of R. rickettsii, rOmpA and rOmpB, and use of these as tools to identify the immunodominant T-lymphocyte epitopes. The role of NO in killing intrahepatocytic rickettsiae and of cytokines such as IL- 1Beta and TNF-alpha in converting human endothelial cells into effectors of rickettsicidal activity will be determined. The human immune response to SFG rickettsial infection will be evaluated by in situ RT-PCR for mRNA of iNOS and the cytokines of interest in eschar biopsies. Effort will be focused on the role of CTL-mediated apoptosis of infected endothelium in the final clearance of rickettsiae. Rickettsial antigens recognized by human CTLs will be identified. Of crucial importance to the development of effective vaccines against rickettsiae and other intracellular parasites are our experiments designed to determine the differential immune components activated by living and killed rickettsiae and by subunit recombinant polypeptides and recombinant BCG.